PROJECT SUMMARY Both establishment of latency by Epstein?Barr virus (EBV) and the virus? ability to reactivate are prerequisites for EBV-associated diseases including B cell- and epithelial cell- derived malignancies. Yet, large gaps in knowledge about the host cell-intrinsic factors that regulate the establishment and maintenance of EBV latency and the life cycle of EBV overall exist. Among the host innate immune/intrinsic mechanisms that are critical for controlling virus replication are several RNA surveillance pathways, most prominently the ones initiated by intracellular RNA sensors, such as RIG-I-like receptors, that induce interferon-mediated antiviral responses. Another important eukaryotic RNA surveillance pathway is the nonsense-mediated mRNA decay (NMD) pathway which recognizes and rapidly degrades certain RNAs. Whereas the role of the NMD machinery in the regulation of cellular processes has been well defined, significantly less is known about the relevance of NMD-mediated RNA decay in controlling virus replication. Intriguingly, a series of recent studies demonstrated that the NMD pathway is critical for restricting the replication of several RNA viruses; however, whether NMD-mediated RNA decay plays a role in controlling the life cycle of DNA viruses, and in particular gamma- herpesviruses such as EBV, is currently unknown. The long-term goal of this study is to understand the physiological relevance of the NMD RNA surveillance machinery in controlling the EBV life cycle (Aim 1). We will utilize molecular and biochemical assays combined with next-generation RNA sequencing to determine the precise RNAs targeted by the NMD machinery in EBV-infected cells, and further elucidate the role of degradation of these RNAs in controlling EBV latent infection and reactivation in various relevant cell types (Aim 2). Finally, we will determine the role of NMD-mediated RNA surveillance in EBV-induced oncogenesis (Aim 3). The research proposed in this application is innovative because it investigates the role of a novel intrinsic host mechanism in EBV infection control and EBV-induced tumorigenesis. Furthermore, the proposed studies are important as they will significantly expand our knowledge about host regulation of EBV infection and likely guide the design of novel therapeutic strategies.